PROJECT SUMMARY Over 30 million men suffer from erectile dysfunction in the U.S. Constriction and dilation of the cavernosal vasculature determines penile erection. In the absence of arousal stimuli, activation of heterotrimeric G proteins following ligand binding to membrane receptors maintains constriction of the cavernosal arterioles and sinuses, keeping the penis non-erect. Subsequent to heterotrimeric G protein activation, two signaling pathways are brought into play to cause constriction: the well-characterized Ca2+-dependent pathway (phospholipase C) and the recently identified RhoA/Rho-kinase pathway known as Ca2+ sensitization. The coupling of G protein receptors to phospholipase C activation is well characterized, but much less is known about how RhoA/Rho-kinase is coupled to G protein activation. Recent evidence indicates that the RhoA activation is positively regulated by Rho guanine nucleotide exchange factors (RhoGEFs). The regulation of RhoGEF activity is also unclear. Interestingly, one particular RhoGEF, PDZ-RhoGEF is regulated by a proline- rich tyrosine kinase 2 (PYK2), a Ca2+-dependent enzyme. We have obtained preliminary data to support a role for PYK2 in vascular responses to angiotensin II. We hypothesize that angiotensin II activation of PYK2 and PDZ-RhoGEF leads to increased RhoA/Rho-kinase to maintain the non-erect state. Further, -term over- expression of PYK2 and PDZ-RhoGEF leads to erectile dysfunction. These hypotheses will be tested by 3 specific aims: 1A) to demonstrate that activation of PYK2/PDZ-RhoGEF leads to vasoconstriction and penile flaccidity;1B) to determine if PYK2 expression in the penis can be modulated to alter erectile function and if siRNA for PDZ-RhoGEF will inhibit components of the RhoA/Rho-kinase signaling pathway;2) to determine if increased PYK2/PDZ-RhoGEF activity contributes to erectile dysfunction in angiotensin II-induced hypertension;and 3) to determine if increased PYK2/PDZ-RhoGEF activity contributes to erectile dysfunction in diabetes. The approach will utilize rat and mouse models of erection. The experiments will determine the biochemical, pharmacological and physiological measures of PYK2/RhoGEF activity with respect to stimulation of the RhoA/Rho-kinase pathway in the intact penis and in isolated cavernosal strips. Gene transfer to over- express PYK2 should lead to a new model of erectile dysfunction whereas;siRNA for PDZ-RhoGEF will reduce signaling via the RhoA/Rho-kinase pathway. Contractile force measurements in isolated cavernosal strips (intact and permeabilized) will provide evidence for Ca2+ sensitization and its regulation by PYK2/PDZ- RhoGEF. Erectile dysfunction induced by diabetes and hypertension will be used to evaluate a possible clinical significance of this signaling pathway. PYK2 (-/-) mice will provide an important approach to studying the PYK2/PDZ-RhoGEF signaling pathway. If supported these studies would provide an explanation for the molecular basis for smooth muscle contraction in the normal state and how long-term changes in the penis contribute to erectile dysfunction. The work will provide insight into novel therapeutic approaches. PROJECT NARRATIVE Over 30 million men suffer from erectile dysfunction in the U.S. Whereas, penile erection is determined by cell signaling mechanisms promoting both constriction and dilation of the cavernosal vasculature, current therapeutic approaches focus on stimulating the dilatory response. Our research has identified novel cellular events regulating constrictor responses, and an understanding of how these events can be inhibited will provide an alternative therapeutic approach to treating erectile dysfunction.